Monoclonal antibody for predicting tamoxifen response in breast cancer patients

ABSTRACT

Aspects of the present invention provide a monoclonal antibody specific for BQ323636.1 and its use for prediction of tamoxifen resistance in breast cancer patients.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/756,824, filed Mar. 1, 2018, which is the U.S. national stageapplication of International Patent Application No. PCT/CN2016/097131,filed Aug. 29, 2016, which claims the benefit of U.S. ProvisionalApplication No. 62/213,661, filed Sep. 3, 2015, the disclosures of eachof which are incorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

Breast cancer is the most common type of malignancy affecting womenworldwide. Estrogen receptor (ER) signaling pathway is a fundamentalpro-proliferative pathway in the context of breast cancer. Upon itsactivation by binding with estrogen, ER activates target genetranscription and cell growth either directly through its genomicpathway or indirectly through non-genomic pathway that involves thePI3K/AKT pathway.

About 70% of breast cancer patients express estrogen receptor alpha(ERα) and can be treated with endocrine therapy. Tamoxifen is aselective estrogen receptor modulator (SERM) that acts as antagonist ofestrogen in the context of breast and is the most commonly prescribedanti-estrogen drug to ER+ patients for prevention of breast cancerrelapse or metastasis. Binding of tamoxifen to ER triggers therecruitment of nuclear co-repressor 2 (NCOR2, also known as SMRT) aswell as other co-repressors such as GPS2, TBLR1, HDAC3 etc., andsuppresses pro-proliferative ER signaling pathway (Sharma, Saxena et al.2006, Zhang, Chang et al. 2006, Cheng and Kao 2009). Despite therelative safe and significant anti-neoplastic activities of tamoxifen ininhibiting disease relapse and improving patients' survival, resistanceis an outstanding issue with up to 50% of non-responding patients, andmany of the initial responders experience relapse (Ring and Dowsett2004).

The mechanisms underlying tamoxifen resistance have been extensivelystudied but not yet well understood. Resistance might arise from one ormore of the factors mentioned: (a) aberrant tamoxifen metabolism thataffects its bioavailability; (b) dysregulation of growth factor receptorpathways and their downstream targets; (c) altered expression/functionof co-regulators; (d) loss of estrogen receptor expression or function;etc.

There has been no available robust biomarker to predict the responsetowards tamoxifen treatment in breast cancer patients. Therefore, almostall the patients with positive ERα status will be prescribed withtamoxifen. Some of patients are resistant to this drug, but by the timethe clinicians realize that the drug has failed, the cancer has alreadyspread and metastasized.

In 2006, the US Food and Drug Administration (FDA) recommended includinginformation on CYP2D6 genotypes and their potential effect on patientoutcomes in the label for tamoxifen, but a consensus on whethergenotyping should be required or considered optional was not reached (deSouza and Olopade 2011). Since then, many more clinical studiesaddressing the relationship between CYP2D6 and tamoxifen resistance havebeen published, but the results have been inconsistent and conflicting.CYP2D6 is a metabolic enzyme that catalyzes 4-hydroxylation of tamoxifen(Dehal and Kupfer 1997, Coller, Krebsfaenger et al. 2002). In terms ofmortality, some studies showed that the CYP2D6 genotype is associatedwith shorter relapse free survival and disease-free survival whileothers could not find such associations (Dezentje, Guchelaar et al.2009, Hoskins, Carey et al. 2009). Another fourteen studies have beenreported on the association between CYP2D6 and the disease recurrence,but the majority of them did not find statistically significantrelationships. Therefore, there is not enough solid data to justify theimplementation of individual CYP2D6 genotyping for adjuvant treatment ofbreast cancer, and it is still questioned whether CYP2D6 can serve as arobust biomarker for predicting tamoxifen resistance.

BRIEF SUMMARY OF THE INVENTION

The present invention relates generally to monoclonal antibodiesspecific to a splice variant of NCOR2, named BQ323636.1, BQ323636.1having the sequence of SEQ ID NO: 1, and their use as predictive fortamoxifen response in estrogen receptor positive breast cancer patients.

In one aspect, the present invention provides an antibody that binds asplice variant of NCOR2, wherein the splice variant is characterized byan exon 11 deletion during mRNA splicing, resulting in an earlytranslation stop codon and truncated protein product. The splice varianthas the sequence of SEQ ID NO: 1.

In some embodiments, the antibody binds an epitope of NCOR2 with thesequence of QRTWRSRCASWP (SEQ ID NO: 2). In some embodiments, theantibody is a chimeric, human, or humanized antibody. In someembodiments, the antibody is an antibody fragment selected from Fab,Fab′, Fab′-SH, F (ab′) 2, Fv, diabodies, single-chain antibody fragment,or a multispecific antibody comprising multiple different antibodyfragments. In some embodiments, the antibody is conjugated or covalentlybound to a detectable moiety.

In another aspect, the present invention provides an antibody obtainedby chimerizing or humanizing an antibody that binds a splice variant ofNCOR2, wherein the splice variant is characterized by an exon 11deletion during mRNA splicing resulting in an early translation stopcodon and truncated protein product.

In another aspect, the present invention provides a kit comprising theantibody that binds a splice variant of NCOR2, wherein the splicevariant is characterized by an exon 11 deletion during mRNA splicingresulting in an early translation stop codon and truncated proteinproduct, optionally further comprising a labeled secondary antibody thatspecifically recognizes the antibody of any one of the above claims.

In another aspect, the present invention provides a hybridoma orrecombinant host cell producing an antibody that binds a splice variantof NCOR2, wherein the splice variant is characterized by an exon 11deletion during mRNA splicing resulting in an early translation stopcodon and truncated protein product.

In another aspect, the present invention provides methods of determiningtamoxifen resistance in subjects with cancer, comprising:

(a) obtaining a tumor sample from the subject;

(b) determining in the sample a level of expression for a splice variantof NCOR2, wherein the splice variant is characterized by an exon 11deletion during mRNA splicing that results in an early translation stopcodon and truncated protein product;

(c) comparing the expression level in (b) to a level of expression in anormal control,

wherein overexpression of the splice variant of NCOR2, with respect tothe control, indicates that the cancer is tamoxifen resistant. In someembodiments, the splice variant has the sequence of SEQ ID NO: 1.

In some embodiments, the comparing step of the methods of the presentinvention comprises contacting the sample with an antibody thatspecifically recognizes an epitope of NCOR2 with the sequence of SEQ IDNO: 2; and detecting the complex between the antibody and the splicevariant of NCOR2. In some embodiments, the splice variant of NCOR2 iscontacted with an antibody that specifically recognizes the splicevariant of NCOR2 in an immunoassay selected from the group consisting ofradioimmunoassay, western blot assay, immunofluorescent assay, enzymeimmunoassay, immunoprecipitation, chemiluminescent assay,immunohistochemical assay, dot blot assay, and slot blot assay.

In another aspect, the present invention provides methods of determiningwhether a tamoxifen-treated subject is at risk of cancer relapse, or atrisk for metastasis, comprising:

(a) obtaining a tumor sample from the subject;

(b) determining in the sample a level of expression for a splice variantof NCOR2 having the sequence of SEQ ID NO: 1, wherein the splice variantis characterized by an exon 11 deletion during mRNA splicing resultingin an early translation stop codon and truncated protein product;

(c) comparing the expression level in (b) to a level of expression in anormal control,

wherein overexpression of the splice variant of NCOR2, with respect tothe control, indicates that the subject is at risk of cancer relapse, oris at risk for metastasis.

In some embodiments of the methods provided, the subject is a human. Insome embodiments of the methods provided, the cancer is breast cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A shows the full peptide sequence of BQ323636.1 (SEQ ID NO: 1)highlighting the epitope (SEQ ID NO: 2) of BQ323636.1-specificmonoclonal antibody in red. FIG. 1B shows a graph of the four clones(C1, D12, G4, H8) with highest titer of antigen specific antibodyproduction selected with ELISA applied to assess the antigen-antibodyspecific binding.

FIGS. 2A-2D show the four clones of the anti-BQ323636.1 antibody thatwere tested in the application of (FIG. 2A) western blot, (FIG. 2B)immuno-precipitation, (FIG. 2C) immunofluorescence staining and (FIG.2D) immunohistochemistry staining. Among the four clones tested, D12 andG4 showed the best performance.

FIGS. 3A-3B indicate that (FIG. 3A) BQ323636.1 was expressed at higherlevel in tamoxifen resistant cell lines AK47 and LCC2 and (FIG. 3B)ectopic over-expression of BQ323636.1 induces tamoxifen resistance.

FIGS. 4A-4B indicate that BQ323636.1 overexpression confers resistanceto tamoxifen in vivo. (FIG. 4A) The control cell lines, ZR75-vector andMCF7-vector, which are originally tamoxifen sensitive, responded well totamxoifen treatment. (FIG. 4B) Both BQ323636.1 over-expressing celllines, ZR75-BQ32363.61 and MCF7-BQ323636.1, were resistant to thetamoxifen treatment, as there was no change of tumor growth comparedbetween treatment and control group.

FIGS. 5A-5B indicate the predictive values of BQ323636.1 for tamoxifenresistance using immunohistochemical staining on tissue microarrayconstructed from 355 cases of archived breast cancer patients' paraffinblocks. For patients who have received tamoxifen treatment, (FIG. 5A)Nuclear BQ323636.1 overexpression is significantly associated withtamoxifen resistance (defined as patients who received tamoxifentreatment and subsequently developed disease relapse or metastasis) byChi-square test (p=3.90×10⁻⁶). (FIG. 5B) Nuclear BQ323636.1 wassignificantly higher in patients who were later found to be tamoxifenresistant (Mann-Whitney U Rank test, p=4.02×10⁻⁶).

FIGS. 6A-6B show that, (FIG. 6A) nuclear BQ323636.1 overexpression wassignificantly correlated with disease relapse (Chi-square test,p=3.47×10⁻⁴); (FIG. 6B) Nuclear BQ323636.1 was significantly higher inpatients who later developed disease relapse (Maim-Whitney U Rank test,p=3.54×10⁻⁴).

FIGS. 7A-7B show that, (FIG. 7A) nuclear BQ323636.1 overexpression wassignificantly correlated with cancer metastasis (Chi-square test,p=1.72×10⁻⁶); (FIG. 7B) Nuclear BQ323636.1 was significantly higher inpatients who later developed metastasis (Mann-Whitney U Rank test,p=1.78×10⁻⁶).

FIGS. 8A-8C indicate the value of BQ323636.1 as a prognostic maker usingimmunohistochemical staining by Kaplan-Meier estimate. (FIG. 8A) Sampledistribution for each clinical parameters. (FIG. 8B) Nuclear BQ323636.1overexpression was significantly associated with poorer overall survival(Log-rank test, p=6.28×10⁻⁵). (FIG. 8C) Nuclear BQ323636.1overexpression was significantly associated with poorer disease-specificsurvival (Log-rank test, p=1.31×10⁻⁴)

FIGS. 9A-9B indicate the value of BQ323636.1 as a prognostic markerusing immunohistochemical staining by cox-regression analysis. (FIG. 9A)Nuclear BQ323636.1 overexpression was significantly associated withpoorer overall survival (risk ratio=1.842, p=0.000) on univariateanalysis, which remained significant on multivariate analysis (riskratio=2.41, p=0.000). (FIG. 9B) Nuclear BQ323636.1 overexpression wassignificantly associated with poorer disease-specific survival (riskratio=2.10, p=0.000) on univariate analysis, which also remainedsignificant on multivariate analysis (risk ratio=3.2, p=0.000).

FIGS. 10A-10B show cell lines that were stably overexpressing BQ323636.1((FIG. 10A) ZR75-BQ323636.1 and (FIG. 10B) MCF7-BQ323636.1) generated bylentiviral-based system. Transfection efficiency was monitored by theGFP signal. Overexpression of BQ323636.1 was further confirmed bywestern blot and qPCR.

FIG. 11 shows PCR results several combinations of Ig variable domainprimers.

FIG. 12 shows V_(H) amino acid sequence alignment for the monoclonalantibody of the present invention. The sequence alignment shows V_(H)amino acid sequences of the heavy chains of: VI-12.1 (SEQ ID NO: 32),VH2.5 (SEQ ID NO: 33), VH 2.2 (SEQ ID NO: 34), VH 2.3 (SEQ ID NO: 35),VH2.6 (SEQ ID NO: 36), and the consensus V_(II) amino acid sequence (SEQID NO: 3).

FIG. 13 shows V_(L) amino acid sequence alignment for the monoclonalantibody of the present invention. The sequence alignment shows V_(L)amino acid sequences of the light chains of: VK2 (2).2 (SEQ ID NO: 38),VK2 (2).3 (SEQ ID NO: 39), VK2 (2).5 (SEQ ID NO: 40), VK2 (2).6 (SEQ IDNO: 41), VK2.1 (SEQ ID NO: 42), VK2.2 (SEQ ID NO: 43), and the consensusV_(L) amino acid sequence (SEQ ID NO: 44).

FIG. 14 shows a graphical representation of the variable domain and CDRloops for the heavy chain (SEQ ID NO: 3) of the monoclonal antibody ofthe present invention.

FIG. 15 shows a graphical representation of the variable domain and CDRloops for the light chain (SEQ ID NO: 44) of the monoclonal antibody ofthe present invention.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is the amino acid sequence of the BQ323636.1 splice variantof NCOR2.

SEQ ID NO: 2 is the amino acid sequence of the region of the BQ323636.1splice variant of NCOR2 bound by the antibodies of the presentinvention.

SEQ ID NO: 3 is the V_(H) consensus amino acid sequence for themonoclonal antibody of the present invention.

SEQ ID NO: 4 is the amino acid sequence for VH2.1.

SEQ ID NO: 5 is the amino acid sequence for VH2.2.

SEQ ID NO: 6 is the amino acid sequence for VH2.3.

SEQ ID NO: 7 is the amino acid sequence for VH2.5.

SEQ ID NO: 8 is the amino acid sequence for VH2.6.

SEQ ID NO: 9 is the nucleic acid sequence for VH2.1.

SEQ ID NO: 10 is the nucleic acid sequence for VH2.2.

SEQ ID NO: 11 is the nucleic acid sequence for VH2.3.

SEQ ID NO: 12 is the nucleic acid sequence for VH2.5.

SEQ ID NO: 13 is the nucleic acid sequence for VH2.6.

SEQ ID NO: 14 is the amino acid sequence for VK2.1.

SEQ ID NO: 15 is the amino acid sequence for VK2.2.

SEQ ID NO: 16 is the amino acid sequence for VK2(2).2.

SEQ ID NO: 17 is the amino acid sequence for VK2(2).3.

SEQ ID NO: 18 is the amino acid sequence for VK2(2).5.

SEQ ID NO: 19 is the amino acid sequence for VK2(2).6.

SEQ ID NO: 20 is the nucleic acid sequence for VK2.1.

SEQ ID NO: 21 is the nucleic acid sequence for VK2.2.

SEQ ID NO: 22 is the nucleic acid sequence for VK2(2).2.

SEQ ID NO: 23 is the nucleic acid sequence for VK2(2).3.

SEQ ID NO: 24 is the nucleic acid sequence for VK2(2).5.

SEQ ID NO: 25 is the nucleic acid sequence for VK2(2).6.

SEQ ID NO: 26 is the amino acid sequence of the last 16 amino acids ofthe V_(L) consensus amino acid sequence SEQ ID NO: 44 for the monoclonalantibody of the present invention.

SEQ ID NO: 27 is the sequence of hCDR1 of the V_(H) of a monoclonalantibody against the peptide of SEQ ID NO: 2.

SEQ II) NO: 28 is the sequence of hCDR2 of the V_(II) of a monoclonalantibody against the peptide of SEQ ID NO: 2.

SEQ ID NO: 29 is the sequence of hCDR3 of the V_(H) of a monoclonalantibody against the peptide of SEQ ID NO: 2.

SEQ ID NO: 30 is the sequence of lCDR1 of the V_(L) of a monoclonalantibody against the peptide of SEQ ID NO: 2.

SEQ ID NO: 31 is the sequence of lCDR3 of the V_(L) of a monoclonalantibody against the peptide of SEQ ID NO: 2.

SEQ ID NO: 32 is the V_(H) amino acid sequence for VH2.1.

SEQ ID NO: 33 is the V_(H) amino acid sequence for VH2.5.

SEQ ID NO: 34 is the V_(H) amino acid sequence for VH2.2.

SEQ ID NO: 35 is the V_(H) amino acid sequence for VH2.3.

SEQ ID NO: 36 is the V_(H) amino acid sequence for VH2.6.

SEQ ID NO: 37 is the consensus V_(H) amino acid sequence.

SEQ ID NO: 38 is the V_(L) amino acid sequence for VK2 (2).2.

SEQ ID NO: 39 is the V_(L) amino acid sequence for VK2 (2).3.

SEQ ID NO: 40 is the V_(L) amino acid sequence for VK2 (2).5.

SEQ ID NO: 41 is the V_(L) amino acid sequence for VK2 (2).6.

SEQ ID NO: 42 is the V_(L) amino acid sequence for VK2.1.

SEQ ID NO: 43 is the V_(L) amino acid sequence for VK2.2.

SEQ ID NO: 44 is the consensus V_(L) amino acid sequence.

DETAILED DESCRIPTION OF THE INVENTION

Cell culture D12 has been deposited on Jul. 2, 2015, with the LeibnizInstitute DSMZ-German Collection of Microorganisms and Cell Cultures,located at Inhoffenstr. 7B, D-38124 Braunschweig, Germany, underconditions that assure that access to the cultures will be availableduring the pendency of this patent application to one determined by theCommissioner of Patents and Trademarks to be entitled thereto under 37CFR 1.14 and 35 U.S.C. 122. The deposit has been assigned accessionnumber DSM ACC3272 and will be available as required by foreign patentlaws in countries wherein counterparts of the subject application, orits progeny, are filed. However, it should be understood that theavailability of a deposit does not constitute a license to practice thesubject invention in derogation of patent rights granted by governmentalaction.

Further, the subject deposit will be stored and made available to thepublic in accord with the provisions of the Budapest Treaty for theDeposit of Microorganisms, i.e., it will be stored with all the carenecessary to keep it viable and uncontaminated for a period of at leastfive years after the most recent request for the furnishing of a sampleof the deposit, and in any case, for a period of at least thirty (30)years after the date of deposit or for the enforceable life of anypatent which may issue disclosing the culture. The depositoracknowledges the duty to replace the deposit should the depository beunable to furnish a sample when requested, due to the condition of thedeposit. All restrictions on the availability to the public of thesubject culture deposit will be irrevocably removed upon the granting ofa patent disclosing it.

Applicants have previously identified a splice variant of NCOR2 (NCOR2is also known as SMRT), named BQ323636.1, which is associated withtamoxifen resistance in breast cancer (Zhang L, Gong C, et al, 2013).The BQ323636.1 splice variant is characterized by skipped exon 11 duringmRNA splicing, resulting in an early translation stop codon and atruncated protein product retaining only the N-terminal fragmentcompared to its wild-type (Zhang, Gong et al. 2013).

The present invention provides an antibody specific for BQ323636.1. Thepresent invention also provides methods involving the use of monoclonalBQ323636.1 antibody for predicting patients' responses to tamoxifentreatment. The development of such a reliable biomarker enablesappropriate alternative therapy to be given to breast cancer patients atan early stage without suffering from undesirable side effects oftamoxifen. Predicting which patients will respond to tamoxifen and whichwill not provide the advantageous ability to provide the correcttreatment early to improve disease outcome.

In one aspect, the present invention provides an antibody that binds asplice variant of NCOR2 having the sequence of SEQ ID NO: 1, wherein thesplice variant is characterized by an exon 11 deletion during mRNAsplicing resulting in an early translation stop codon and truncatedprotein product.

In some embodiments, the antibody binds an epitope of NCOR2 with thesequence of SEQ ID NO: 2. In some embodiments, the antibody is achimeric, human, or humanized antibody. In some embodiments, theantibody is an antibody fragment selected from Fab, Fab′, F (ab′) 2, Fv,diabodies, single-chain antibody fragment, or a multispecific antibodycomprising multiple different antibody fragments. In some embodiments,the antibody is conjugated or covalently bound to a detectable moiety.

In another aspect, the present invention provides an antibody obtainedby chimerizing or humanizing an antibody that binds a splice variant ofNCOR2, wherein the splice variant is characterized by an exon 11deletion during mRNA splicing resulting in an early translation stopcodon and truncated protein product. In some embodiments, the splicevariant has the sequence of SEQ ID NO: 1. In some embodiment, theantibody binds an epitope on the NCOR2 splice variant having thesequence of SEQ ID NO: 2.

The term “antibody” may be used interchangeably with the term“immunoglobulin” and is defined herein as a protein synthesized by ananimal or a cell of the immune system in response to an antigen orimmunogen. The term antibody as used herein also refers to fragmentsthereof. Antibodies are characterized by specific affinity to a site onthe antigen referred to as an “antigenic determinant” or an “epitope”.Antigens can be naturally occurring or artificially engineered. Theimmunoglobulin molecules utilized in the present invention can be of anytype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgA1, IgA2,IgG1, IgG2, IgG3, and IgG4) or subclass of immunoglobulin molecule.

Antibodies can include either polyclonal or monoclonal antibodies. Inpreferred embodiments, the antibodies of the present invention aremonoclonal antibodies. In addition to intact immunoglobulin molecules,also included are fragments or polymers of those immunoglobulinmolecules, and human or humanized versions of immunoglobulin moleculesor fragments thereof, as long as the molecules maintain the ability tobind with an epitope of the NCOR2 splice variant. The antibodies can betested for their desired activity using the in vitro assays describedherein, or by analogous methods, after which the in vivo therapeuticand/or diagnostic activities can be confirmed and quantified accordingto known clinical testing methods.

The antibodies utilized in the present invention are inclusive of allspecies, and the antigenic target can be from any species. Mostpreferably, the antibodies are human antigen-binding antibodies, andfragments thereof, and include, but are not limited to, Fab, Fab′ andF(ab′)2, Fd, single-chain Fvs (scFv), single-chain antibodies,disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VHdomain. Antigen-binding antibody fragments, including single-chainantibodies, may comprise the variable region(s) alone or in combinationwith the entirety or a portion of the hinge region, CH1, CH2, and/or CH3domains. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Also included in the inventionare antigen-binding fragments comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and/or CH3 domains.

The antibodies and aptamers useful in the present invention may begenerated by any suitable method known in the art. Monoclonal antibodiescan be prepared using any of the techniques known in the art, includingthe use of hybridoma, recombinant, and phage display technologies, or acombination thereof. The term “monoclonal antibody” or “mAb” as usedherein is not limited to antibodies produced through hybridomatechnology. The term refers to any antibody that is derived from asingle clone, including any eukaryotic, prokaryotic, or phage clone, andnot the method by which it is produced. Methods for producing andscreening for specific antibodies using hybridoma technology are routineand well known in the art. Similarly, methods for producing andscreening for specific aptamers are routine and well known in the art.

In some instances, it may be desirable to use chimeric, humanized, orhuman antibodies in the invention. A chimeric antibody is a molecule inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art and mayinclude splicing genes from a mouse antibody molecule of appropriateantigen specificity together with genes from a human antibody moleculeof appropriate biological activity. Humanized antibodies are antibodymolecules from a non-human species antibody that binds the desiredantigen having one or more complementarity determining regions (CDRs)from the non-human species and a framework region from a humanimmunoglobulin molecule. Completely human antibodies may be desirablefor therapeutic treatment of human patients. Human antibodies can bemade by a variety of methods known in the art.

In some embodiments, the antibody of the present invention has a V_(H)amino acid sequence of SEQ ID NO:3 and/or a V_(L) amino acid sequence ofSEQ ID NO: 44.

In another aspect, the present invention provides a kit comprising theantibody or aptamer that binds a splice variant of NCOR2, wherein thesplice variant is characterized by an exon 11 deletion during mRNAsplicing resulting in an early translation stop codon and truncatedprotein product, optionally further comprising a labeled secondaryantibody or aptamer that specifically recognizes the anti-variant-NCOR2antibody or aptamer. The splice variant comprises the sequence of SEQ IDNO: 1.

In another aspect, the present invention provides a hybridoma orrecombinant host cell producing an antibody that binds a splice variantof NCOR2, wherein the splice variant is characterized by an exon 11deletion during mRNA splicing resulting in an early translation stopcodon and truncated protein product. The splice variant comprises thesequence of SEQ ID NO: 1.

In another aspect, the present invention provides methods of determiningtamoxifen resistance in subjects with cancer, comprising:

(a) obtaining a tumor sample from a subject;

(b) determining in the sample a level of expression for a splice variantof NCOR2, wherein the splice variant is characterized by an exon 11deletion during mRNA splicing resulting in an early translation stopcodon and truncated protein product;

(c) comparing the expression level in (b) to a level of expression in anormal control,

wherein overexpression of the splice variant of NCOR2, with respect tothe control, indicates that the cancer is tamoxifen resistant. In someembodiments, the splice variant has the sequence of SEQ ID NO: 1.

In some embodiments, the comparing step of the methods of the presentinvention comprises contacting the sample with an antibody or aptamerthat specifically recognizes an epitope of NCOR2 with the sequence ofSEQ ID NO: 2; and detecting the complex between the antibody or aptamerand the splice variant of NCOR2. In some embodiments, the splice variantof NCOR2 is contacted with an antibody that specifically recognizes thesplice variant of NCOR2 in an immunoassay selected from the groupconsisting of radioimmunoassay, western blot assay, immunofluorescentassay, enzyme immunoassay, immunoprecipitation, chemiluminescent assay,immunohistochemical assay, dot blot assay, and slot blot assay.

In another aspect, the present invention provides methods of determiningwhether a tamoxifen-treated subject is at risk of cancer relapse, or atrisk for metastasis, comprising:

(a) obtaining a tumor sample from the subject;

(b) determining in the sample a level of expression for a splice variantof NCOR2 having the sequence of SEQ ID NO: 1, wherein the splice variantis characterized by an exon 11 deletion during mRNA splicing resultingin an early translation stop codon and truncated protein product;

(c) comparing the expression level in (b) to a level of expression in anormal control,

wherein overexpression of the splice variant of NCOR2, with respect tothe control, indicates that the subject is at risk of cancer relapse, oris at risk for metastasis.

In some embodiments of the methods provided, the subject is a human. Insome embodiments of the methods provided, the cancer is breast cancer.

The antibodies, or antibody fragments thereof, and aptamers particularlyuseful in the present invention specifically bind to an epitopecomprising the amino acid sequence set forth in SEQ ID NO: 2.

Without further elaboration, it is believed that one skilled in the artcan, using the preceding description, utilize the present invention toits fullest extent. The following examples are offered by way ofillustration, not by way of limitation. While specific examples havebeen provided, the above description is illustrative and notrestrictive. Anyone or more of the features of the previously describedembodiments can be combined in any manner with one or more features ofany other embodiments in the present invention. Furthermore, manyvariations of the invention will become apparent to those skilled in theart upon review of the specification.

All publications and patent documents cited in this application areincorporated by reference in pertinent part for all purposes to the sameextent as if each individual publication or patent document were soindividually denoted. By citation of various references in thisdocument, Applicants do not admit any particular reference is “priorart” to their invention.

EXAMPLES

The methods, antibodies, and kits herein described are furtherillustrated in the following examples, which are provided by way ofillustration and are not intended to be limiting. It will be appreciatedthat variations in proportions and alternatives in elements of thecomponents shown will be apparent to those skilled in the art and arewithin the scope of embodiments of the present invention. Theoreticalaspects are presented with the understanding that Applicants do not seekto be bound by the theory presented. All parts or amounts, unlessotherwise specified, are by weight.

The following materials and methods were used for all the methods,antibodies, and kits exemplified herein.

Cell Lines—

Human breast cancer cell lines MCF7 and ZR-75-1 were purchased fromAmerican Type Culture Collection and were re-authenticated by shorttandem repeat profiling (Zhang, Gong et al. 2013). MCF7 was cultured inDulbecco's Modified Eagle's Medium supplemented with 10% FBS and 1%penicillin/streptomycin and ZR-75-1 was cultured in Improved MinimumEssential Medium (IMEM) supplemented with 5% FBS and 1%penicillin/streptomycin. LCC2 and AK-47 are the two tamoxifen resistantcell lines derived from MCF7 and ZR-75-1, respectively and were kindlyprovided by Dr. Robert Clarke (Georgetown University Medical School,Washington, D.C.) (Wong, Dai et al. 2006) and have been used in ourprevious study (Zhang, Gong et al. 2013). LCC2 and AK-47 were bothcultured in IMEM supplemented with 5% charcoal-stripped FBS and 1%penicillin/streptomycin. All the cell lines used in this study have beenpassaged and kept fewer than 6 months after the re-authentication orthawing.

Lentiviral Transfection for Generation of Stable Cell Line—

For lentivirus production, early passage 293FT cells were plated at5×10⁵ cells/well in 6-well plates two days before transfection. Thecells were grown to 80-90% confluency to perform transfection.Recombinant lentiviruses were produced by co-transfecting 293FT cellswith the lentivirus expression plasmid and packaging plasmids usingLipofectamine 2000 (Invitrogen). 10 μl of Lipofectamine 2000 reagent wasdiluted in 250 μl plain medium and incubated at 5 minutes at roomtemperature. 1 μg of lentivirus expression plasmid and 2.6 μl pPACKPackaging Plasmid Mix (System Biosciences) was added to the medium withdiluted Lipofectamine 2000 reagent. While this was further incubated atroom temperature for 15 minutes, fresh complete medium was replaced.After the 20 minutes incubation, the DNA-Lipofectamine complexes wereadded to the cells and incubated overnight. On the next day, the mediumwith the complexes were discarded and 1 ml fresh medium containing 30%(v/v) FBS was added. At the 48 hours post transfection, the medium washarvested and centrifuged at 2000 rpm for 5 minutes at room temperatureto remove cell debris. The viral supernatant was saved and stored at−80° C. until use. Target cells to be transfected were plated at3-5×10⁵/well in 6-well plates 24-48 hours prior to viral infection. Onthe day of transduction, the medium was removed and replaced with lmlfresh complete medium with Polybrene (Sigma) at a final concentration of12 μg/ml. The target cells were infected by adding 2000-8000 preparedviral supernatant. After 24 hours, the culture medium was discarded andreplaced with 1 ml complete medium without Polybrene. The infectedtarget cells could be selected by adding complete medium containing0.5-1 ug/ml puromycin (Sigma) 3-7 days post infection. GFP expressionwas examined by fluorescent microscopy after 3-4 days.

MTT Assay—

Cells were seeded at a density of 6×10³ cells/well. On the day ofconducting MTT assay, cells were incubated with culture mediumcontaining 10% MTT (USB, Affymetrix, stock concentration is 5 mg/mL) at37° C. for 4 hours. After incubation, the medium was discarded andreplaced with 100 mL isopropanol with 4 mmol/L HCl and 0.1% NP-40 todissolve the purple precipitates. Extinction of the samples was measuredby the Infinite 200 microtiter plate reader (Tecan) at 570 nm with areference wavelength at 750 nm. Background MTT readings were subtractedfrom the samples.

Immunoprecipitation—

Cells were lysed in IP lysis buffer (0.025M Tris, 0.15M NaCl, 0.001MEDTA, 1% NP-40, 5% glycerol, pH 7.4) and pre-cleared with 30 μl ofDynabeads Protein A/G (A for rabbit antibodies, G for mouse antibodies,Invitrogen, Life Technologies, UK) by rotating at 4° C. for 4 hours.After pre-clearing, protein concentration was measured and the lysatewas split into equal amount of proteins per tube and incubated with thespecific primary antibody or IgG negative control (dilution 1:200) at 4°C. for 0/N with gentle rotating. On the second day, 400 of beads wereadded to the mixture and incubated at 4° C. for another 4 h. Afterincubation, the beads were washed five times with cold PBS and boiled at100° C. for 5 min to elute the proteins. Proteins were separated bySDS-PAGE gel electrophoresis, transferred to nitrocellulose membrane andhybridized with the antibodies same as Western blotting.

Immunofluorescence Staining—

Briefly, cells were fixed with 50:50 methanol:acetone (Millipore,Germany/Merck, UK) for 10 min at −20° C. Samples were then incubatedovernight with the primary mouse anti-BQ (clone D12) which we havegenerated. Following washes with PBS, secondary goat anti-mouse IgG-FITC(1:2000, Life technologies, US) was added to the samples for an hour at37° C. incubator. Cells were mounted with mounting medium with DAPI(Life technologies, US). Images were captured and quantified using theCarl Zeiss LSM 710 confocal laser scanning microscope and softwareWindows Vista, Z E N 2011 version 5.5 SP1.

Orthotopic Mouse Model—

The cell lines mixed with Matrigel (BD Biosciences) at the ratio of 1:1were inoculated into the abdominal mammary fat pad of the 5 to 6 weekold female nude mice. When the tumor were palpable, the mice wererandomized into treatment and control groups where treatment groupsreceived daily subcutaneous injection of tamoxifen (Sigma) dissolved inpeanut oil (Sigma) and the control groups received subcutaneousinjection of the solvent only. Tumor sizes were measured by caliper andtumor volume was calculated as (length*width*width)/2. Tumor growth ratewas expressed as the % of tumor volume change calculated as the tumorvolume measured at the indicated time point normalized against the tumorvolume at day 0*100%. This protocol has been reviewed and approved byCommittee on the Use of Live Animals in Teaching and Research (CULATR),the University of Hong Kong (CULATR No.: 3259-14)

Tissue Microarray—

Two hundred and thirty-six cases of breast cancer diagnosed between theyears 1992 to 2001 with clinical follow up data were retrieved from therecords of the Department of Pathology, Queen Mary Hospital of HongKong, with approval by the Institutional Review Board of The Universityof Hong Kong (UW 06-379 T/1404). Histological sections of all cases werereviewed by the pathologist, the representative paraffin tumour blockschosen as donor block for each case and the selected areas marked forconstruction of tissue microarray (TMA) blocks. A total of 206 casescould be assessed and scored for BQ323636.1 staining. Of these 93 hadreceived tamoxifen treatment and were ER positive.

We further recruited for analysis TMA sections provided by ourcollaborators from Nottingham University Hospital. This consisted of alarge cohort of patients comprising a well-characterised consecutiveseries of early stage (TNM Stage I-III excluding T3 and T4 tumours)sporadic primary operable invasive breast cancers from patients (age ≤70years) enrolled into the Nottingham Tenovus Primary Breast CarcinomaSeries that presented at Nottingham City Hospital between 1989 and 1998and managed in accordance to a uniform protocol. The study was approvedby the Nottingham Research Ethics Committee 2 under the title‘Development of a molecular genetic classification of breast cancer’.The TMA sections used included 1129 breast cancer patients withpathological and clinical follow up data of over 20 years. A total of679 cases could be assessed and scored for BQ323636.1 staining. Of these262 had received tamoxifen treatment and were ER positive.

Hence a total of 355 Tamoxifen treated ER positive breast cancer casesfrom Hong Kong and the UK were used for statistical analysis.

Immunohistochemistry—

The TMA sections were deparaffinized and rehydrated by incubation withxylene and decreasing concentrations of ethanol. Citrate buffer (0.01M,pH 6.0) was used for antigen retrieval. The slides were immersed into 3%H₂O₂/methanol for 10 min at room temperature to quench endogenousperoxidase. After rinsing in 0.05% Tween in PBS (PBST) twice, BQ323636.1specific antibody diluted at 1:50 was added to each section andincubated at 4° C. overnight. The slides were then washed in PBST andincubated with DAKO EnVision+System-HRP-labelled Polymer Anti-Rabbit atroom temperature in dark for 30 min. After washing, ChromogenDAB/substrate reagent was added onto the slides and the slides incubatedfor a further 6 minutes. Finally, the slides were dehydrated andmounted. Aperio ScanScope® system (Aperio technology, USA) was used tovisualize and assess for BQ323636.1 expression. The TMA slides werescanned by ScanScope scanners and individual stained TMA spots wereassessed in computer screen with the use of Aperio's image viewer,ImageScope. To avoid subjectivity in evaluation, the intensities andpercentages of the staining were scored by two independent individualsin a semi-quantitative way as previously described and average wastaken. BQ323636.1 expression level was scored according to the H-scoresystem which takes into account the intensity of the staining andpercentage of each intensity (Detre, Saclani Jotti et al. 1995). Hscore=(1×% of cells stained at intensity category 1)+(2×% of cellsstained at intensity category 2)+(3×% of cells stained at intensitycategory 3). The cutoff was set as the median of the scores, which was130.

Statistical Analysis—

Statistical analyses were conducted used SPSS (IBM, version 17). Resultsfrom MTT assays and mouse model were compared by students' t-test. Thecorrelations between expression levels of splice variant BQ323636.1 andtamoxifen responsiveness of the patients were analyzed by Chi-squaretests. The expression levels of BQ323636.1 were compared betweendifferent groups using Mann-Whiney U Rank test. Survival analyses weredone by Kaplan-Meier estimates and Cox regression model. P values ofless than 0.05 were considered statistically significant.

Example 1: Generation of Monoclonal Antibody Specific to BQ323636.1

The present invention involves the generation of a mouse monoclonalantibody whose epitope (QRTWRSRCASWP) (SEQ ID NO: 2) mapped to the last11 amino acids of the BQ323636.1 protein (FIG. 1A), which is thesequence which distinguishes BQ323636.1 from its wild-type NCOR2/SMRT.The detailed protocol is described below.

Immunogen: Peptide 3781.1 Biosyntan GmbH QRTWRSRCASWP-OHPeptide-BSA-Conjugate (BSA: Fraction V, Pierce; Cross-Linker: Sulfo-MBS,Pierce)

Host: 8-week-old female BALB/c mice

Immunization procedures:

Immunisation Doses (Day) Injection (μg/mouse) Adjuvant 0 Priming 109TiterMax Gold Adjuvant (i.p.) 14 Boost 1 109 Complete Freund's (i.p.)Adjuvant 21 Boost 2 54 Incomplete Freund's (i.p.) Adjuvant 28 Boost 3 54Incomplete Freund's (i.p.) Adjuvant 35 Boost 4 54 PBS (i.p.) 38 FusionMouse 42

The presence of the desired antibody was detected in the sera of therecipients prior to the final booster dose using the enzyme-linkedimmunosorbent assay (ELISA) with the uncoupled Peptide 3781 asimmobilized antigen. For fusion experiment mouse 42 was used.

Date of Fusion:

Apr. 10, 2013 Fusion number: 577

Parent Cell Fusion Line:

SP2/0 (non-secreting mouse myeloma)

Fusion Method:

Two days before fusion mouse (Balb/c) peritoneal macrophages for use asfeeder cells were prepared and seeded into the wells of four 96-wellcell culture plates. For fusion procedures, 6×10⁷ spleen cells from theimmunised mouse and 2×10⁷ cells from mouse myeloma cell line SP2/0 wereincubated with 1.2 ml of polyethylene glycol 1450 (50% in 10% DMSO;Sigma) for 30 sec. at 37° C. After washing, the cells were seeded in thefour 96-well cell culture plates. Hybrid clones were selected by growingin HAT medium [RPMI 1640 culture medium (Biochrom) supplemented with 20%fetal calf serum (PAN) and HATSupplement (50×; PAN)]. After two weeksthe HAT medium was replaced with HT Medium for three passages followedby returning to the normal cell culture medium.

Screening/Cloning/Recloning:

The cell culture supernatants were primary screened for antigen specificIgG antibodies two weeks after fusion. The presence of antigen specificantibodies in the culture supernatants was measured by its binding tothe uncoupled Peptide 3781 attached directly to the wells of 96-wellmicrotiter plates (100 ng/well). The antibody binding was quantified byadding the relevant anti-species immunoglobulin to which an enzyme isbound, followed by a chromogenic substrate to that enzyme. Fresh culturemedium and a dilution of the polyclonal mouse antiserum were used asnegative or as positive controls. 12 specific antibody producinghybridoma colonies were found to be high positive. The selected 12 cellpopulations were transferred into 24-well plates for cell propagationand were then tested again. 4 cell populations with the highestantibodies titres were selected for cloning and re-cloning using thelimiting-dilution technique. 4 cell lines producing specific monoclonalantibodies were characterized and frozen. For the isotypecharacterization the Mouse Monoclonal Antibody Isotyping Kit (Roche) wasused.

Cell Culture Medium:

RPMI 1640 Medium [(1×), w 2.0 g/l NaHCO₃, w stable Glutamine (BiochromAG; Catalog No.: FG 1415)] with 20% FCS (PAN Biotech GmbH; Cat.-No.:1302-P283004; Lot No.: P283004; Origin: Australia); Gentamicin (PAALaboratories GmbH; Best.-Nr. P11-004; 50 μg/ml)

Optimal Growth:

At a temperature of 37° C. and 5% CO2

Storage of Cells/Freezing:

Approximately 3×10⁶ cells in 750 μl fresh cell culture medium werepipette into a cryotube. 750 μl of freezing medium (80% FCS and 20% DMSO(PAN)) were added. Using a Nalgene Cryo Freezing Container the cryotubeswere placed in a −80° C. freezer immediately and transferred to liquidN2 within 24 hours for long-term storage.

Storage of cells/Thawing: The cryotubes were quickly thawed as possiblein a 37° C. water bath. The cells were diluted with 10 ml cold mediumand centrifuged at 1000 rpm for 10 min. Then the pellets wereresuspended in cell culture medium and transferred into 25 ml cellculture flasks.

Test for Mycoplasma:

The cell culture supernatants were tested for Mycoplasma using an ELISAKit (Mycoplasma detection Kit; Roche Diagnostics GmbH)

Example 2: Reactivities of the Secreted Antibodies of the 4 FinalClones: ELISA

The cell culture supernatants were incubated with a solid phase to whichthe uncoupled Peptide 3781 has been bound by direct adsorption. Duringthe assay any specific antibody will itself become bound to the antigenon the solid phase, and then in a second incubation is detected with alabelled anti-species antibody.

Coating: 100 ng/well (well=50 μl) of Peptide 3781 (diluted in PBS)overnight at 4° C.; wash plate twice with wash buffer (PBS/0.05%Tween20). Blocking: 100 ul/well blocking solution (PBS/0.05% Tween20/10%NCS) for 1 h at RT;

Wash plates twice with wash buffer. Incubation: 50 ul/well of the cellculture supernatants serial diluted in incubation buffer (PBS/0.05%;Tween20/10% NCS) for 2 h at RT; wash plates three times with washbuffer. Conjugate: 50 ul/well HRP-Rabbit anti Mouse IgG; (Fc specific;Pierce; diluted in incubation buffer) for 1 h at RT; wash plates threetimes with wash buffer. Substrate: 50 μl/well enzyme substrate (OPD incitrate-hydrogenphosphate buffer) for 15 min at RT. Stopping: 50 μl/wellstopping solution. Measure: Measure absorbance at 490 nm

Substrate: 3.7 mM o-Phenylendiamine in Citrate/Hydrogenphosphate buffer,0.012% H2O2 Citrate/Hydrogenphosphate buffer: 35 mM Citric acidMonohydrate, 85 mM Na2HPO4×2H2O, pH 5.0 Stopping solution: 4NSchwefelsäure. Wash buffer: PBS/0.05% Tween20.

Four clones (C1, D12, G4, H8) with highest titer of antigen specificantibody production were selected and ELISA was applied to assess theantigen-antibody specific binding (FIG. 1B).

Example 3: Testing of Anti-BQ323636.1 Antibody in Commonly UsedBiomedical Research Methods

The four clones of the anti-BQ323636.1 were tested in the application ofwestern blot (FIG. 2A), immuno-precipitation (FIG. 2B),immunofluorescence staining (FIG. 2C) and immunohistochemistry staining(FIG. 2D). In western blot, this BQ323636.1 detects a specific band atmolecular weight of slightly below 50 kDa (predicted molecular weight is42.65 kDa, antibody dilution 1:1000). Immuno-precipitation by clone G4and D12 showed enrichment of the protein at molecular weight slightlybelow 50 kDa. Immunofluorescence staining (antibody dilution 1:50)showed that BQ323636.1 was expressed in both cytoplasm and nucleus ofthe cell, consistent with immunohistomchemistry staining using breastcancer patients' samples (antibody dilution 1:50).

Example 4: BQ323636.1 Overexpression Conferred Tamoxifen Resistance BothIn Vitro and in Vivo

Western blot using anti-BQ323636.1 antibody showed that, at proteinlevel, BQ323636.1 expressed at higher level in the derived tamoxifenresistant cell lines LCC2 and AK47 compared to their parental tamoxifensensitive cell lines MCF7 and ZR75, respectively (FIG. 3A). Usinglentiviral-based system, we generated cell lines that were stablyoverexpressing BQ323636.1 (ZR75-BQ323636.1 and MCF7-BQ323636.1) (FIGS.10A-10B) and cell lines transfected with empty vector were used as thecontrol (ZR75-vector and MCF7-vector). MTT assays showed that BQ323636.1overexpression induced the tamoxifen resistance, as shown in FIG. 3B.

We further validated the effect of BQ323636.1 overexpression inconferring tamoxifen resistance using a nude mouse model. The cell linesmixed with Matrigel (BD Biosciences) at the ratio of 1:1 were inoculatedinto the abdominal mammary fat pad of the 5 to 6 week old female nudemice. When the tumors were palpable, the mice were randomized intotreatment and control groups where treatment groups received dailysubcutaneous injection of tamoxifen (Sigma) dissolved in peanut oil(Sigma) and the control groups received equivalent amount of solventonly. Tumor sizes were measured by caliper and tumor volume wascalculated as (length*width*width)/2. Tumor growth rate was expressed asthe % of tumor volume change calculated as the tumor volume measured atthe indicated time point normalized against the tumor volume at day0*100%. As shown in FIG. 4A, the control cell lines, ZR75-vector andMCF7-vector, which are originally tamoxifen sensitive, responded well totamxoifen treatment, as marked by the significantly reduced tumor growthwhen compared with the control group which had received solventtreatment only. In contrast, BQ323636.1 over-expressing cell lines, bothZR75-BQ32363.61 and MCF7-BQ323636.1 (FIG. 4B), were resistant to thetamoxifen treatment, as there was no change of tumor growth comparedbetween treatment and control group. Both the in vitro and in vivoresults confirmed that BQ323636.1 overexpression conferred tamoxifenresistance, which provide solid experimental evidence for the use ofBQ323636.1 as a predictive marker for tamoxifen resistance.

Example 5: Use of Monoclonal Antibody Anti-BQ323636.1 in THC forPrediction of Tamoxifen Resistance in Breast Cancer Patients

As in vivo confirmation of the usefulness of BQ323636.1 as a predictivemarker for tamoxifen resistance in human tissue samples, the efficacy ofusing monoclonal antibody specific for BQ323636.1 was assessed by IHCstaining on tissue microarray on a total of 355 patients who hadreceived tamoxifen treatment. These tamoxifen treated patients were fromHong Kong, (93 cases) and from the United Kingdom (262 cases). Bothcohorts of patients when analyzed separately, each gave statisticallysignificant results. Hence the pooled results are presented. Tamoxifenresistance was defined as patients who received tamoxifen treatment andsubsequently developed disease relapse or metastasis.

Nuclear BQ323636.1 overexpression was significantly associated withtamoxifen resistance by Chi-square test (p=3.90×10⁻⁶, FIG. 5A) andBQ32363.1 nuclear expression was significantly higher in patients whowere later found to be tamoxifen resistant (Mann-Whitney U Rank test,p=4.02×10⁻⁶, FIG. 5B). Nuclear BQ323636.1 overexpression was alsosignificantly associated with disease relapse (Chi-square test,p=3.47×10⁻⁴, FIG. 6A) and nuclear BQ323636.1 was significantly higher inpatients who later developed disease relapse (Mann-Whitney U Rank test,p=3.54×10⁻⁴, FIG. 6B). Moreover, nuclear BQ323636.1 overexpression wassignificantly correlated with cancer metastasis (Chi-square test,p=1.72×10⁻⁶, FIG. 7A) and nuclear BQ323636.1 was significantly higher inpatients who later developed metastasis (Mann-Whitney U Rank test,p=1.78×10⁻⁶, FIG. 7B).

Consistent with its role in predicting tamoxifen resistance, nuclearBQ323636.1 overexpression was significantly associated with poorersurvival by Kaplan-Meier estimate (Log-rank test, p=6.28×10⁻⁵ andp=1.31×10⁻⁴ for overall survival and disease-specific survival,respectively, FIGS. 8A-8C). By cox regression univariate analysis (FIGS.9A-9B), it was also found that nuclear BQ323636.1 overexpression wassignificantly associated with poorer overall survival (risk ratio=1.842,p=0.000) as well as with poorer disease-specific survival (riskratio=2.10, p=0.000). The significance was maintained on multivariateanalysis with the association with poorer overall survival (riskratio=2.41, p=0.000) as well as with poorer disease-specific survival(risk ratio=3.20, p=0.000).

Example 6: Sequencing of V_(H) and V_(L) Chains of the MonoclonalAntibody

mRNA was extracted from the hybridoma cell pellets. Total RNA wasextracted from the pellets using Fusion Antibodies Ltd in-house RNAextraction protocol.

RT-PCR:

cDNA was created from the RNA by reverse-transcription with an oligo(dT)primer. PCR reactions are set up using variable domain primers toamplify both the V_(H) and V_(L) regions of the monoclonal antibody DNAgiving the bands shown in FIG. 11. The V_(H) and V_(L) products werecloned into the Invitrogen sequencing vector pCR2.1 and transformed intoTOP10 cells and screened by PCR for positive transformants. Selectedcolonies were picked and analyzed by DNA sequencing on an AB13130xlGenetic Analyzer. Amino acid sequence alignments are shown for the heavychain in FIG. 12 and the light chain in FIG. 13.

The V_(H) Consensus Amino Acid Sequence:

(SEQ ID NO: 3) MYLGLSCVFIVFLLKGVQSEVKLEESGGGLVQPGGSMKLSCVAS GFT F SN YW(hCDR1)MNWVRQSPEKGLEWVAE IRLRSSYYAT (hCDR2)HYAESVKGRFTISRDDSKSSVYLQMNNLRAEDTGIYYC TMITTGYEDV (hCDR3)WGGATTVTVSSAKTTPPSVYPLAThe variable domain is highlighted in BOLD. The ComplementarityDetermining Regions (CDRs) are underlined as determined by the IMGTnumbering system (Lefranc, M. P. et al., Nucleic Acids Research, 27,209-212 (1999)).

A graphical representation of the variable domain and the CDR loops forthe heavy chain is shown in FIG. 14.

The amino acid sequences aligned in FIG. 12 include VH2.1 (SEQ ID NO:32), VH2.2 (SEQ ID NO: 34), VH2.3 (SEQ ID NO: 35), VH2.5 (SEQ ID NO:33), and VH2.6 (SEQ ID NO: 36), having corresponding nucleic acidsequences of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,and SEQ ID NO: 13, respectively.

V_(L) Consensus Amino Acid Sequence:

(SEQ ID NO: 44) MKLPVRLLVLMFWIPASSSDVVMTQTPLSLPVSLGDQASISCRSS QSLIHSNGNTY(ICDR1) LHWYLQKPGQSPKWY KVS(ICDR2) NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFC SQITHIPRT(ICDR3) FGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKThe variable domain is highlighted in BOLD. The ComplementarityDetermining Regions (CDRs) are underlined as determined by the IMGTnumbering system (Lefranc, M. P. et al., Nucleic Acids Research, 27,209-212 (1999)).

A graphical representation of the variable domain and the CDR loops forthe heavy chain is shown in FIG. 15.

The amino acid sequences aligned in FIG. 13 include VK2.1 (SEQ ID NO:42), VK2.2 (SEQ ID NO: 43), VK2(2).2 (SEQ ID NO: 38), VK2(2).3 (SEQ IDNO: 39), and VK2(2).5 (SEQ ID NO: 40), and VK2(2).6 (SEQ ID NO: 41),having corresponding nucleic acid sequences of SEQ ID NO: 20, SEQ ID NO:21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25,respectively.

DISCUSSION

The first front treatment for breast cancer patients is usually surgicalresection. More than 70% of breast cancers express estrogen receptor(ER). ER positive patents are routinely treated with tamoxifen, aselective estrogen receptor modulator, as first-line adjuvant therapy toinhibit cancer recurrence and reduce mortality rate. However, withprolonged tamoxifen treatment, almost half of patients will eventuallydevelop resistance and present with disease recurrence or metastases.These patients will then need to be treated with chemotherapy and willgenerally do poorly.

Since about one-half of the breast patients with estrogenreceptor-positive cancer fail on tamoxifen, identification of effectiveand reliable biomarkers that could be used to monitor tamoxifen efficacyand new targets to reverse tamoxifen resistance is of importance. Inorder to improve the disease outcome of breast cancer patients,establishment of more sensitive predictive biomarkers and noveltherapeutic targets for patients who are refractory to tamoxifentreatment is needed. The present invention provides a monoclonalantibody specific for BQ323636.1 and the use of it in assays, such asimmunohistochemistry (IHC), for prediction of tamoxifen resistance inbreast cancer patients. BQ323636.1, a splice variant of NCOR2/SMRT, wasidentified to be associated with tamoxifen resistance and a mousemonoclonal antibody specific for this splice variant was generated tostudy the efficacy of using BQ323636.1 as a predictive marker fortamoxifen resistance. Using this antibody, it was found thatoverexpression of BQ323636.1 conferred resistance to tamoxifen both invitro using cell lines and in vivo using nude mice model. Using thisantibody in IHC on tissue microarrays (TMA) constructed from 355 casesof archived breast cancer patients' paraffin blocks successfully showedthat BQ323636.1 overexpression could predict tamoxifen resistance(p=3.90×10⁻⁶) (tamoxifen resistance being defined as patients whoreceived tamoxifen and later developed relapse or metastasis) and wasassociated with poor patients' survival (p=6.28×10⁻⁵ for overallsurvival, p=1.31×10⁻⁴ for disease specific survival).

REFERENCES

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What is claimed is:
 1. An antibody or antibody fragment thereof thatspecifically binds a splice variant of NCOR2, wherein the splice variantis characterized by an exon 11 deletion during mRNA splicing resultingin an early translation stop codon and truncated protein product.
 2. Theantibody or antibody fragment of claim 1, wherein the antibody orantibody fragment specifically binds an epitope of NCOR2 consisting ofthe sequence of SEQ ID NO:
 2. 3. The antibody or antibody fragment ofclaim 1, wherein the antibody is a chimeric, human, or humanizedantibody.
 4. The antibody fragment of claim 1, wherein the antibodyfragment is selected from Fab, Fab′, Fab′-SH, F (ab′) 2, Fv, diabodies,single-chain antibody fragment, and a multispecific antibody comprisingmultiple different antibody fragments.
 5. The antibody or antibodyfragment of claim 1, wherein said antibody or antibody fragment isconjugated or covalently bound to a detectable moiety.
 6. An antibodyobtained by chimerizing or humanizing an antibody of claim
 1. 7. Theantibody or antibody fragment of claim 1, wherein the antibody orantibody fragment is a monoclonal antibody.
 8. The antibody or antibodyfragment of claim 1, wherein the antibody or antibody fragment has aV_(H) amino acid sequence of SEQ ID NO:
 3. 9. The antibody or antibodyfragment of claim 1, wherein the antibody or antibody fragment has aV_(L) amino acid sequence of SEQ ID NO:
 26. 10. A kit comprising theantibody or antibody fragment of claim 1, the kit optionally furthercomprising a labeled secondary antibody that specifically recognizes theantibody or antibody fragment of claim
 1. 11. A hybridoma or recombinanthost cell producing the antibody or antibody fragment of claim
 1. 12. Amethod of determining tamoxifen resistance in a subject with cancer andtreating the subject, the method comprising: (a) obtaining a tumorsample from the subject; (b) determining in the sample a level ofexpression for a splice variant of NCOR2, wherein the splice variant ischaracterized by an exon 11 deletion during mRNA splicing resulting inan early translation stop codon and truncated protein product, whereinsaid determining the expression level comprises using the antibody orantibody fragment of claim 1; and (c) comparing the expression level in(b) to a level of expression in a normal control, wherein overexpressionof the splice variant of NCOR2, with respect to the control, indicatesthat the cancer is tamoxifen resistant; and d) treating the subject withchemotherapy if the cancer is tamoxifen resistant.
 13. The method ofclaim 12, wherein the level of expression in the normal control is belowan H-Score of 130 and overexpression is a level of expression above anII-Score of
 130. 14. The method of claim 12, wherein the subject is ahuman.
 15. The method of claim 12, wherein the cancer is breast cancer.16. The method of claim 15, wherein the comparing step comprises:contacting the sample with the antibody or antibody fragment thatspecifically recognizes an epitope of NCOR2 consisting of the sequenceof SEQ ID NO: 2; and detecting the complex between the antibody orantibody fragment and the splice variant of NCOR2.
 17. The methodaccording to claim 16, wherein the splice variant of NCOR2 is contactedwith the antibody or antibody fragment that specifically recognizes thesplice variant of NCOR2 in an immunoassay selected from the groupconsisting of radioimmunoassay, western blot assay, immunofluorescentassay, enzyme immunoassay, immunoprecipitation, chemiluminescent assay,immunohistochemical assay, dot blot assay, and slot blot assay.
 18. Amethod of determining whether a tamoxifen-treated subject is at risk ofcancer relapse, or at risk for metastasis, and treating the subject ifthe subject is at risk of cancer relapse or at risk for metastasis, themethod comprising: (a) obtaining a tumor sample from thetamoxifen-treated subject; (b) determining in the sample a level ofexpression for a splice variant of NCOR2, wherein the splice variant ischaracterized by an exon 11 deletion during mRNA splicing resulting inan early translation stop codon and truncated protein wherein saiddetermining the level of expression comprises using the antibody orantibody fragment of claim 1; and (c) comparing the expression level in(b) to a level of expression in a normal control, wherein overexpressionof the splice variant of NCOR2, with respect to the control, indicatesthat the tamoxifen-treated subject is at risk of cancer relapse or is atrisk for metastasis; and d) treating the subject with chemotherapy ifthe cancer is at risk of cancer relapse or at risk for metastasis. 19.The method of claim 18, wherein the subject is a human.
 20. The methodof claim 19, wherein the cancer is breast cancer.